利用脂質體學探討兩種淋巴細胞株暴露游離輻射後之生物效應

Abstract

游離輻射為可直接或間接使物質產生游離作用之電磁輻射或粒子輻射，具有相當高的反應性並會對生物體造成軀體傷害或是導致癌症。然而游離輻射生物效應機制至今尚未解析。近年來，體學技術如基因體學、轉錄體學與代謝體學蓬勃發展，提供高通量分子量測方法研究游離輻射所產生之健康效應。 輻射暴露相關研究中p53基因為最常被探討的基因之一，當p53基因功能不全時，細胞受游離輻射暴露後將無法正常地進行細胞凋亡作為保護機制而增加突變機率。代謝體為生物反應中的最終產物，能更直接反映生物體內因基因改變或環境差異而產生的變化。脂質由於其結構多樣性及其富含之生化意義，因此可從代謝體學中獨立為脂質體學，其中Glycerophosphocholines （GPCs）與Sphingomyelins （SMs）為細胞膜上含量最多的脂質，同時也具有多種生物意義。過去研究指出脂質為游離輻射產生之自由基的主要攻擊標的物，為此我們使用液相層析儀串聯質譜儀之分析方法分析GPCs 與SMs。 本研究使用人類淋巴原母細胞株WTK1 （p53 mutant-type）細胞進行游離輻射劑量效應暴露實驗，於暴露後3小時以及24小時收集樣本；同時也比較WTK1細胞株與TK6 （p53 wild-type）細胞株於未暴露狀態下脂質體之差異。以UPLC-MS/MS平台取得GPCs和SMs圖譜訊號，經圖譜前處理後進行多變量分析以及統計檢定。 結果顯示WTK1劑量效應組別中發現Alkenyl-acyl PCs在細胞內的量會隨暴露劑量增加而增加，推測Alkenyl-acyl PCs扮演抗氧化劑的角色以保護細胞免於活性氧化物質 （Reactive oxygen species, ROS）攻擊；而GPCs在細胞中量的增加則能調節細胞膜的張力，改變碳鏈之不飽和度以減少細胞膜氧化作用發生，降低膜通透性並促進細胞進行修復。雖於本研究中未觀察到WTK1細胞進行細胞凋亡，然而SMs濃度的減少可能與水解為Ceramide作為細胞凋亡傳訊分子相關，飽和GPCs濃度下降等現象，暗示來自輻射暴露之氧化攻擊仍會讓細胞走向凋亡。而比較TK6與WTK1兩種細胞株脂質體顯示其含量已有差異。 本研究顯示GPCs在細胞內的含量會受p53基因狀態而改變，當p53基因突變之細胞暴露輻射後除了影響細胞週期也會導致GPCs為保護細胞而產生變化。未來GPCs與SMs可發展作為輻射誘導毒性之潛在生物標誌。

Ionizing radiation is one type of radiations that can liberate an electron from an atom or molecule. In other words, ionizing radiation is highly reactive and able to cause cancer or other serious problems to living organisms. Though we already know the radiation damage in the body, the mechanisms of those biological effects are still unclear. Recent “omics” technologies such as genomics, transcrptomics and metabolomics have been flourished which can prvide high through-put molecular measurement to examine effects of genetic modulation or environmental effects in biological samples. p53 gene is the most often discussed genes which involved in radiation exposure studies. Radiated cells will increase mutation frequency when p53 gene is dysfunction and may not process apoptosis as protect mechanism. Lipidomics can assist comprehensive understanding of lipids which are the end products of cellular processes and able to directly reflect changes of biological function. Glycerophosphocholines (GPCs) and sphingomyelins (SMs), which were the most abundant lipids on cell membrane, also have various biological functions. The profiling of those lipids may be changes after radiation exposure. Since that, we use an established liquid chromatography-tandem MS method to analyze them. In this study, we use human lymphoblastic cell lines WTK1 (mutant p53) to examine lipidomic effects of ionizing radiation. Dose-response experiments were conducted in cells treated with gamma-ray (iso-survival dose, D0 (TK6: 0.8 Gy; WTK1: 1.5 Gy) or 10 Gy) for 3 or 24 hours. At the same time we compare the GPC and SM profiling of WTK1 and TK6 (wild-type p53) cell lines when not exposed to ionizing radiation. GPCs and SMs are analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometer (UPLC-MS/MS) following multivariate statistical anlayis. Results show that in WTK1 cell lines dose-response group, the increased alkenyl-acyl PCs can act as antioxidant to protect cell from reactive oxygen species (ROS). The increase of GPCs in cell can modulate membrane tension, reduce membrane oxidation by change the degree of unsaturation, and lower membrane permeability to process cell membrane repair after the attack of oxidative stress from radiation exposure. Though we did not observe cell apoptosis, our results show the decrease of SMs after radiation exposure may be related to apoptosis since SMs may be hydrolyzed to ceramide as a signal messenger, while the decrease of saturated GPCs might also cause further apoptosis. There’s also shown lipid abundance variation between two cell lines. This study suggest the GPCs level within the cell may be alterd due to the difference of p53. Not only the cell cycle was alterd but also the changes of GPCs were performed to protect cell after radiation exposure. This made GPCs and SMs may serve as potential markers for irradiation-induce toxicity.